The sigma-1 receptor (Sig-1R) has emerged as a significant target in the realm of pain management and has been the subject of extensive research. Nonetheless, its specific function in inflammatory pain within dorsal root ganglion (DRG) neurons remains inadequately elucidated. This study utilized whole-cell patch clamp techniques, single-cell real-time PCR, and immunohistochemistry to examine the influence of Sig-1R on inflammatory pain induced by complete Freund's adjuvant (CFA) in a rat model. Our results revealed several key findings: 1) The expression of Sig-1R was found to be upregulated during the progression of inflammatory pain, with a notable translocation from the cytoplasm to the membrane; 2) Inhibition of peripheral Sig-1R using S1RA resulted in a reduction of CFA-induced allodynia; 3) Activation of Sig-1R through PRE-084 led to a decrease in the fast sodium current in isolated DRG neurons from CFA-treated rats, which was associated with a diminished action potential (AP) peak and maximum depolarizing rate (MDR), as well as an increased rheobase; 4) Furthermore, PRE-084 was observed to enhance the slow component of the sodium current, resulting in hyperpolarization of the threshold potential and an increase in AP firing frequency, alongside an elevation in the mRNA expression of the slow sodium channel Nav1.9 in CFA-treated rats. In conclusion, our findings suggest that the modulation of sodium channels by Sig-1R in DRG neurons plays a significant role in the mechanisms underlying inflammatory pain.

In vitro experiments performed on dissociated dorsal root ganglion (DRG) neurons suggest the involvement of the hyperpolarization-activated cation current (Ih) in enhancing neuronal excitability, potentially contributing to neuropathic pain. However, the more confirmative in vivo information about how nerve injury interacts with Ih is lacking. In this study, Ih was recorded in vivo using the dynamic single-electrode voltage clamp (dSEVC) technique on L5 DRG neurons of normal rats and those seven days after spinal nerve axotomy (SNA). Compared to normal rats, SNA unexpectedly inhibited the activity of Ih channels on A-fiber DRG neurons: (a) the Ih current magnitude, density, and conductance were consistently diminished; and (b) the Ih activation velocity was slowed and the voltage for Ih activation was hyperpolarized. The half-activation voltage (V0.5) exhibited a negative shift, and the time constant for Ih activation was prolonged across all test potentials, indicating the reduced availability of Ih after SNA. To further investigate the mechanisms of SNA on Ih, the underlying HCN channels and the correlated mRNA were quantified and compared. The mRNA expression level of HCN1-4 was uniformly enhanced after SNA, which might have contributed to the increased cytoplasmic HCN1 intensity observed in both medium- and large-sized DRG neurons. This finding contradicted the functional reduction of Ih after SNA. Surprisingly, the HCN labeling pattern was altered after SNA: the labeling area of HCN1 and HCN2 at the membranous ring region of the axotomized large neurons became significantly thinner or absent. We concluded that the diminished ring immunoreactivity for HCN1 and HCN2 correlated with a reduced availability of Ih channels, elucidating the observed decrease in Ih in axotomized A-fiber neurons.

The A20 ubiquitin-editing enzyme is a critical negative regulator of NF-κB signaling and inflammation. While the mechanisms by which A20 restricts inflammation have been extensively studied, the physiological functions of other A20-like proteins are largely unknown. Here, we report a previously unknown function of the A20 family member ZFAND6 as a novel regulator of mitophagy. Deletion of ZFAND6 in bone marrow-derived macrophages (BMDMs) promotes the upregulation of reactive oxygen species (ROS) and the accumulation of damaged mitochondria due to impaired mitophagy. Consequently, mitochondrial DNA (mtDNA) is released into the cytoplasm, triggering the spontaneous expression of interferon-stimulated genes (ISGs) in a cGAS-STING dependent manner, which leads to enhanced viral resistance in vitro . However, mice lacking ZFAND6 exhibit increased morbidity and mortality upon challenge with a sublethal dose of influenza A virus (IAV) due to impaired myeloid cell activation and diminished type I IFN signaling. Mechanistically, ZFAND6 bridges a TRAF2-cIAP1 interaction, which is required for the initiation of ubiquitin-dependent mitophagy. Our results suggest that ZFAND6 is a subunit of a TRAF2-cIAP E3 ligase complex that promotes the clearance of damaged mitochondria by mitophagy to maintain mitochondrial homeostasis. ### Competing Interest Statement The A.G.-S. laboratory has received research support from GSK, Pfizer, Senhwa Biosciences, Kenall Manufacturing, Blade Therapeutics, Avimex, Johnson & Johnson, Dynavax, 7Hills Pharma, Pharmamar, ImmunityBio, Accurius, Nanocomposix, Hexamer, N-fold LLC, Model Medicines, Atea Pharma, Applied Biological Laboratories, Merck and Castlevax, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: Castlevax, Amovir, Vivaldi Biosciences, Contrafect, 7Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Applied Biological Laboratories, Pharmamar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, Pfizer and Prosetta, outside of the reported work. A.G.-S. has been an invited speaker in meeting events organized by Seqirus, Janssen, Abbott, Astrazeneca and Novavax. A.G.-S. is inventor on patents and patent applications on the use of antivirals and vaccines for the treatment and prevention of virus infections and cancer, owned by the Icahn School of Medicine at Mount Sinai, New York, outside of the reported work. A.P. has consulted for GlaxoSmithKline. The other authors declare no competing interests.

Objective:To evaluate the effects of learning initiative on teaching effectiveness.Methods:The research subjects were the 2nd year medical students of Tongji Medical College of Huazhong University of Science and Technology. The learning initiative factor was calculated from the data of the attendance registration of the on-line learning of Physiology, and the performance of the on-line test was used as an evaluation indicator of teaching effectiveness. SPSS software was used to perform correlation analysis between the learning initiative factor and teaching effectiveness. Results:We found that learning initiative could significantly affect the teaching effectiveness, with differences among different specialties. There was a positive correlation between learning initiative and teaching effectiveness in clinical and pediatric medicine, while no correlation was observed in preventive medicine and medical imaging.Conclusion:In conclusion, learning initiative can affect teaching effectiveness, and the intensity of this effect shows difference among different specialties.

目的 探讨二甲双胍对单侧输尿管梗阻?(UUO)?诱导的小鼠肾脏纤维化的影响及其作用机制.方法 选取30只C57BL/6小鼠,分为假手术组?(仅分离左侧输尿管但不结扎,n?=?10)、UUO组?(单侧输尿管结扎术法建立UUO小鼠模型,n?=?10)、?二甲双胍组[单侧输尿管结扎术法建立UUO小鼠模型,造模前1?d开始二甲双胍灌胃,200?mg/?(kg·d),连续7?d;?n?=?10].HE染色检测3组肾组织形态变化;?Masson染色测定3组肾间质纤维化情况;?免疫组化检测3组肾组织中YAP1、TGF-β1和CD68的表达;?免疫荧光检测3组肾组织中F4/80的表达;?Western?blotting检测YAP1、p-YAP、LATs1、TEAD的表达.结果 与假手术组比较,UUO组尿N-乙酰-β-D-葡萄糖苷酶增加,肾间质纤维化程度增加,CD68、F4/80、TGF-β1、YAP1、TEAD表达增高,而p-YAP、LATs1的表达下降,差异均有统计学意义?(均P?0.05).与UUO组比较,二甲双胍组尿N-乙酰-β-D-葡萄糖苷酶下降,肾间质纤维化程度减轻,CD68、F4/80、TGF-β1、YAP1、TEAD表达减少,而p-YAP、LATs1的表达增加,差异均有统计学意义?(均P?0.05).结论 UUO诱导的肾脏纤维化激活肾组织中Hippo/YAP通路,上调CD68、F4/80、TGF-β1蛋白表达水平促进肾纤维化,引起肾结构及功能异常;?二甲双胍可能通过干预Hippo/YAP通路改善UUO诱导的小鼠肾脏结构及功能的损伤.

The severe doxorubicin (DOXO) side effect of cardiomyopathy limits it clinical application as an effective anticancer drug. Although Ca2+ overload was postulated as one of the mechanisms for this toxicity, its role was, however, disputable in terms of the contractile dysfunction. In this work, the dynamics of the intracellular Ca2+ signal were optically mapped in a Langendorff guinea pig heart. We found that DOXO treatment: (1) Delayed the activation of the Ca2+ signal. With the reference time set at the peak of the action potential (AP), the time lag between the peak of the Ca2+ signal and AP (Ca-AP-Lag) was significantly prolonged. (2) Slowed down the intracellular Ca2+ releasing and sequestering process. Both the maximum rising (MRV) and falling (MFV) velocity of the Ca2+ signal were decreased. (3) Shortened the duration of the Ca2+ signal in one cycle of Ca2+ oscillation. The duration of the Ca2+ signal at 50% amplitude (CaD50) was significantly shortened. These results suggested a reduced level of intracellular Ca2+ after DOXO treatment. Furthermore, we found that the effect of tachypacing was similar to that of DOXO, and, interestingly, DOXO exerted contradictory effects on the tachypaced hearts: it shortened the Ca-AP-Lag, accelerated the MRV and MFV, and prolonged the CaD50. We, therefore, concluded that DOXO had a different effect on intracellular Ca2+. It caused Ca2+ underload in hearts with sinus rhythm; this might relate to the contractile dysfunction in DOXO cardiomyopathy. It led to Ca2+ overload in the tachypaced hearts, which might contribute to the Ca2+-overload-related toxicity.

Systemic lupus erythematosus (SLE) afflicts more than 1.5 million individuals in the United States with very limited and debilitating therapeutic options. The etiology of the disease remains unclear. It is hypothesized that in SLE, peripheral B cell tolerance is breached by inappropriate survival of autoreactive cells. Consistently, overexpression of B cell activating factor (BAFF), a key regulator of B cell survival can rescue autoreactive B cells and contribute to autoimmune disease. BAFF counters apoptosis in part through decreasing BH3-only pro-apoptotic protein Bim. Notably, both BAFF and Bim are physiological regulators of B cell tolerance. Therefore, to define the role of B cells and B cell tolerance in the initiation and progression of autoimmune disease we created a mouse model wherein gene encoding Bim is selectively deleted in B cells (B.Bimf/f). Initial analyses of these mice suggest that B cells can initiate an autoimmune pathogenesis phenotypically similar to SLE, with mice exhibiting splenomegaly and increased CD21loCD23lo B cells. Given that persistent activity of NF-kB in B cells can also result in autoimmune diseases, we are now investigating whether the persistent NF-kB and dysregulated apoptosis can synergize in SLE pathogenesis. To that end, we have crossed B.Bimf/f mice with a mouse line that lacks a recently discovered potential negative regulator of NF-kB, ZFAND6 (B.Bimf/f x ZfanD6−/−). Preliminary findings suggest that the compound mutant mice display exacerbated autoimmune symptoms with higher incidence of splenomegaly and kidney pathology. These preliminary results suggest that a breach in tolerance (B.Bimf/f ) and persistent NF-kB activity cooperate in the initiation and severity of SLE pathogenesis.

The dissociated dorsal root ganglion (DRG) neurons with or without culture were widely used for investigation of their electrophysiological properties. The culture procedures, however, may alter the properties of these neurons and the effects are not clear. In the present study, we recorded the action potentials (AP) and the voltage-gated Na+, K+, and Ca2+ currents with patch clamp technique and measured the mRNA of Nav1.6–1.9 and Cav2.1–2.2 with real-time PCR technique from acutely dissociated and 1-day (1-d) cultured DRG neurons. The effects of the nerve growth factor (NGF) on the expression of Nav1.6–1.9 and Cav2.1–2.2 were evaluated. The neurons were classified as small (DRG-S), medium (DRG-M), and large (DRG-L), according to their size frequency distribution pattern. We found 1-d culture increased the AP size but reduced the excitability, and reduced the voltage-gated Na+ and Ca2+ currents and their corresponding mRNA expression in all types of neurons. The lack of NGF in the culture medium may contribute to the reduced Na+ and Ca2+ current, as the application of NGF recovered some of the reduced transcripts (Nav1.9, Cav2.1, and Cav2.2). 1-d culture showed neuron-type specific effects on some of the AP properties: it increased the maximum AP depolarizing rate (MDR) and hyperpolarized the resting membrane potential (RP) in DRG-M and DRG-L neurons, but slowed the maximum AP repolarizing rate (MRR) in DRG-S neurons. In conclusion, the 1-d cultured neurons had different properties with those of the acutely dissociated neurons, and lack of NGF may contribute to some of these differences.

Myocytes in the pulmonary veins (PV) play a pivotal role in the development of paroxysmal atrial fibrillation (AF). It is therefore important to understand physiological characteristics of these cells. Studies on these cells are, however, markedly impeded by the fact that single PV myocytes are very difficult to obtain due to lack of effective isolation methods. In this study, we described a novel PV myocyte isolation method. The key aspect of this method is to establish a combination of retrograde heart perfusion (via the aorta) and anterograde PV perfusion (via the pulmonary artery). With this simultaneous perfusion method, a better perfusion of the PV myocytes can be obtained. As results, the output and viability of single myocytes isolated by simultaneous heart and PV perfusion method were increased compared with those in conventional retrograde heart perfusion method.

Real-time PCR is a powerful tool for quantifying nucleic acid expression. Real-time PCR is conventionally performed at the tissue level to guarantee an abundance of nucleic acid for detection. The precision and reliability of this method, however, is limited by usually being composed of a mixture of different cell types. Single-cell PCR, in contrast, eliminates the purity problem of the cell source. However, use of this method is usually impeded by difficulties in cell harvesting and stringent requirements for processing of very small quantities of nucleic acids. In this study, we combined the advantages of the high purity of selected cells in single-cell PCR with the greater nucleic acid quantities and thus greater ease of tissue-level PCR. The key aspect of our method is to use a modified patch-clamp pipette to harvest several selected cells of the same type. This method is therefore especially useful for cells that can be morphologically or histologically identified such as primary sensory neurons, striated muscle fibers and cells labeled with fluorescent makers.

Background: Traditional Chinese herbal medicines (TCMs) have been widely used against a broad spectrum of biological activities, including influencing the cardiac differentiation from mouse embryonic stem cells (mESCs). However, their effects and mechanisms of action on ESCs proliferation remain to be determined. The present study aimed to determine the effect of three TCMs, baicalin, ginsenoside Rg1, and puerarin, on mESCs proliferation and to elucidate the possible mechanism of their action. Methods: Cell proliferation was examined with a cell proliferation assay Cell Counting Kit-8 (CCK-8), propidium iodide (PI) staining was used to visualize cell cycle. The mRNA expression level of c-myc, c-fos, c-jun, GAPDH and microRNAs were measured by quantitative real time RT-PCR. Results: We found that baicalin 50 μM suppressed the proliferation of mESCs as observations in more cells in G1 phase and less cells in either S phase or G2/M phase. Moreover, baicalin suppressed the expressions of c-jun and c-fos in mESCs and down-regulated the expression of miR-294. Overexpression of miR-294 in mESCs significantly reversed the effects of baicalin both on mESC proliferation and c-fos/c-jun expression. Conclusions: Baicalin down-regulation of miR-294 may be its key mechanism of action in decreasing mESCs proliferation.

BACKGROUND:The exact molecular mechanisms regarding HTLV-1 Tax-mediated viral gene expression and CD4 T-cell transformation have yet to be fully delineated. Herein, utilizing virus-infected primary CD4+ T cells and the virus-producing cell line, MT-2, we describe the involvement and regulation of Myocyte enhancer factor-2 (specifically MEF-2A) during the course of HTLV-1 infection and associated disease syndrome.RESULTS:Inhibition of MEF-2 expression by shRNA and its activity by HDAC9 led to reduced viral replication and T-cell transformation in correlation with a heightened expression of MEF-2 in ATL patients. Mechanistically, MEF-2 was recruited to the viral promoter (LTR, long terminal repeat) in the context of chromatin, and constituted Tax/CREB transcriptional complex via direct binding to the HTLV-1 LTR. Furthermore, an increase in MEF-2 expression was observed upon infection in an extent similar to CREB (known Tax-interacting transcription factor), and HATs (p300, CBP, and p/CAF). Confocal imaging confirmed MEF-2 co-localization with Tax and these proteins were also shown to interact by co-immunoprecipitation. MEF-2 stabilization of Tax/CREB complex was confirmed by a novel promoter-binding assay that highlighted the involvement of NFAT (nuclear factor of activated T cells) in this process via Tax-mediated activation of calcineurin (a calcium-dependent serine-threonine phosphatase). MEF-2-integrated signaling pathways (PI3K/Akt, NF-κB, MAPK, JAK/STAT, and TGF-β) were also activated during HTLV-1 infection of primary CD4+ T cells, possibly regulating MEF-2 activity.CONCLUSIONS:We demonstrate the involvement of MEF-2 in Tax-mediated LTR activation, viral replication, and T-cell transformation in correlation with its heightened expression in ATL patients through direct binding to DNA within the HTLV-1 LTR.

In recent years, the contribution of I(f), an important pacemaker current, and intracellular Ca2+ release (ICR) from sarcoplasmic reticulum to pacemaking and arrhythmia has been intensively studied. However, their functional roles in embryonic heart remain uncertain. Using patch clamp, Ca2+ imaging, and RT-PCR, we found that I(f) regulated the firing rate in early and late stage embryonic ventricular cells, as ivabradine (30 mu M), a specific blocker of I(f), slowed down action potential (AP) frequency. This inhibitory effect was even stronger in late stage cells, though I(f) was down-regulated. In contrast to I(f), ICR was found to be indispensable for the occurrence of APs in ventricular cells of different stages, because abolishment of ICR with ryanodine and 2-aminoethoxydiphenyl borate (2-APB), specific blockers of ryanodine receptors (RyRs) and inositol trisphosphate receptors (IP3Rs), completely abolished APs. In addition, we noticed that RyR- and IP3R-mediated ICR coexisted in early-stage ventricular cells and RyRs functionally dominated. While at late stage RyRs, but not IP3Rs, mediated ICR. In both early and late stage ventricular cells, Na-Ca exchanger current (INa/Ca) mediated ICR-triggered depolarization of membrane potential and resulted in the initiation of APs. We also observed that different from I(f), which presented as the substantial component of the earlier diastolic depolarization current, application of ryanodine, and/or 2-APB slowed the late phase of diastolic depolarization. Thus, we conclude that in murine embryonic ventricular cells I(f) regulates firing rate, while RyRs and IP3Rs (early stage) or RyRs (late stage)-mediated ICR determines the occurrence of APs. J. Cell. Biochem. 114: 1852-1862, 2013. (c) 2013 Wiley Periodicals, Inc.

A key feature of the innate antiviral immune response is a rapid nonspecific response to virus infection largely mediated by the induction and extracellular secretion of type I interferons (IFNs) that restrict virus replication. Cytoplasmic sensors such as RIG-I recognize viral RNA and trigger antiviral signaling pathways that upregulate IFN transcription. However, it remains largely unknown how antiviral signaling is negatively regulated to maintain homeostasis after the elimination of virus. In this report, we have identified the RING domain-containing protein RING finger 11 (RNF11) as a novel negative regulator of innate antiviral signaling. Overexpression of RNF11 downregulated IFN-β expression and enhanced viral replication whereas siRNA-mediated knockdown of RNF11 suppressed viral replication. RNF11 interacted with the noncanonical IKK kinases TBK1/IKKi and attenuated their Lys63-linked polyubiquitination by blocking interactions with the E3 ligase TRAF3. The inhibitory function of RNF11 was dependent on the ubiquitin-binding adaptor molecule TAX1BP1 which was required for RNF11 to target TBK1/IKKi. Collectively, these results indicate that RNF11 functions together with TAX1BP1 to restrict antiviral signaling and IFN-β production.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 genome encodes the Tax protein that plays essential regulatory roles in HTLV-1 replication and oncogenic transformation of T lymphocytes. Despite intensive study of Tax, how Tax interfaces with host signaling pathways to regulate virus replication and drive T-cell proliferation and immortalization remains poorly understood. To gain new insight into the mechanisms of Tax function and regulation, we used tandem affinity purification and mass spectrometry to identify novel cellular Tax-interacting proteins. This screen identified heat shock protein 90 (HSP90) as a new binding partner of Tax. The interaction between HSP90 and Tax was validated by coimmunoprecipitation assays, and colocalization between the two proteins was observed by confocal microscopy. Treatment of HTLV-1-transformed cells with the HSP90 inhibitor 17-DMAG elicited proteasomal degradation of Tax in the nuclear matrix with concomitant inhibition of NF-κB and HTLV-1 long terminal repeat (LTR) activation. Knockdown of HSP90 by lentiviral shRNAs similarly provoked a loss of Tax protein in HTLV-1-transformed cells. Finally, treatment of HTLV-1-transformed cell lines with 17-DMAG suppressed HTLV-1 replication and promoted apoptotic cell death. Taken together, our results reveal that Tax is a novel HSP90 client protein and HSP90 inhibitors may exert therapeutic benefits for ATL and HAM/TSP patients.

I(h), which influences neuronal excitability, has recently been measured in vivo in sensory neuron subtypes in dorsal root ganglia (DRGs). However, expression levels of HCN (hyperpolarization-activated cyclic nucleotide-gated) channel proteins that underlie I(h) were unknown. We therefore examined immunostaining of the most abundant isoforms in DRGs, HCN1 and HCN2 in these neuron subtypes. This immunostaining was cytoplasmic and membrane-associated (ring). Ring-staining for both isoforms was in neurofilament-rich A-fiber neurons, but not in small neurofilament-poor C-fiber neurons, although some C-neurons showed cytoplasmic HCN2 staining. We recorded intracellularly from DRG neurons in vivo, determined their sensory properties (nociceptive or low-threshold-mechanoreceptive, LTM) and conduction velocities (CVs). We then injected fluorescent dye enabling subsequent immunostaining. For each dye-injected neuron, ring- and cytoplasmic-immunointensities were determined relative to maximum ring-immunointensity. Both HCN1- and HCN2-ring-immunointensities were positively correlated with CV in both nociceptors and LTMs; they were high in Aβ-nociceptors and Aα/β-LTMs. High HCN1 and HCN2 levels in Aα/β-neurons may, via I(h), influence normal non-painful (e.g. touch and proprioceptive) sensations as well as nociception and pain. HCN2-, not HCN1-, ring-intensities were higher in muscle spindle afferents (MSAs) than in all other neurons. The previously reported very high I(h) in MSAs may relate to their very high HCN2. In normal C-nociceptors, low HCN1 and HCN2 were consistent with their low/undetectable I(h.) In some C-LTMs HCN2-intensities were higher than in C-nociceptors. Together, HCN1 and HCN2 expressions reflect previously reported I(h) magnitudes and properties in neuronal subgroups, suggesting these isoforms underlie I(h) in DRG neurons. Expression of both isoforms was NT3-dependent in cultured DRG neurons. HCN2-immunostaining in small neurons increased 1 day after cutaneous inflammation (CFA-induced) and recovered by 4 days. This could contribute to acute inflammatory pain. HCN2-immunostaining in large neurons decreased 4 days after CFA, when NT3 was decreased in the DRG. Thus HCN2-expression control differs between large and small neurons.